Journal of Gerontology: MEDICAL SCIENCES
2005, Vol. 60A, No. 9, 1212–1218
Copyright 2005 by The Gerontological Society of America
Postexercise Nutrient Intake Enhances Leg Protein
Balance in Early Postmenopausal Women
Lars Holm,1 Birgitte Esmarck,2 Charlotte Suetta,1 Keitaro Matsumoto,3 Tatsuya Doi,3
Masao Mizuno,4 Benjamin F. Miller,1 and Michael Kjaer1
Institute of Sports Medicine, Copenhagen, Bispebjerg Hospital, Denmark.
2
Copenhagen Muscle Research Centre, Rigshospitalet, Denmark.
3
Saga Nutraceuticals Research Institute, Otsuka Pharmaceutical, Saga, Japan.
4
Research Unit 247, Ribe County Hospital Esbjerg, Esbjerg, Denmark.
Background. We investigated the effect of nutrient administration after a session of resistance exercise on muscle
protein kinetics in six healthy, early postmenopausal women, in a crossover design of random and double-blinded
administration of protein and carbohydrate (PC) or placebo (NON).
Methods. Fasted participants received a primed-constant infusion of L-[ring-2H5]-phenylalanine. After 90 minutes of
rest, the participants performed leg-resistance exercises followed by the oral supplementation. During the following
4 hours, net protein balance (NB) and rate of disappearance and appearance of phenylalanine were calculated from
arterial–venous blood samples and blood flow measurements.
Results. NB was elevated ( p , .001) in the PC group compared to the NON group, and NB was not different from zero
in the PC group, whereas it was negative in the NON group. Net balance results were supported by kinetic data from
a reduced number of participants, showing that rate of disappearance was responsible for the initial (,1 hour) effect of PC,
whereas a reduced rate of appearance enhanced the NB from 1.5 to 3 hours after training in the PC group.
Conclusion. In early postmenopausal women, nutrient ingestion following resistance exercise improved anabolism by
enhancing NB in skeletal muscle.
A
DVANCING age leads to a decrease in skeletal muscle
mass (1), which may interfere with life quality and
longevity (2). In general, women have less muscle mass than
men, hence, an accelerated loss of muscle may become
restrictive to their everyday function at an earlier age than
men (3). Thus, middle-aged women are a high-priority target
group for a preventative intervention toward functionrestricting sarcopenia. Middle-aged women also enter
menopause, which is characterized by severe reduction of
female sex hormones. Even though the exact role of the
female sex hormones on muscle and lean body mass remains
uncertain (4,5), many physiological changes are known to
happen during these years, which might make menopausal
women incomparable with women in other age groups.
It is well established that muscle activity, at least in the
form of heavy resistance exercise, improves muscle protein
accretion by elevating protein-synthetic processes more than
protein degradation (6), and that this ability is retained
throughout age (7,8). Similarly, it is clear that protein
retention at rest is enhanced in the postprandial situation
compared with the fasted state (9,10). It is also well described
that young individuals derive an acute anabolic advantage
when combining resistance exercise and nutritional intake
(11–13). However, the muscle protein responsiveness in
middle-aged, postmenopausal women to this combination has
not been investigated. Some studies (9,14,15) indicate that the
nutritional responsiveness may be lower with increasing age.
Further studies are needed to distinguish exercise and
nutrition interactions in an effort to preserve muscle strength,
and thus, functional ability in this susceptible population.
1212
The aim of the present study was to investigate the effect
of protein and carbohydrate ingestion immediately after an
acute resistance exercise session on leg muscle protein
balance and kinetics in early postmenopausal women who
are not using hormone substitution.
METHODS
Participant Selection
Women were recruited from a newspaper advertisement.
During the initial interview, women with known myoskeletal
disorders, frequent use of medication, and history of
resistance exercise training in the year prior to the study
were not invited for further investigation. After an initial
interview, 10 women were invited to a physical examination,
and a 12-hour fasting blood sample, which was screened for
substances (such as immunocytes, electrolytes, creatinine,
enzymes, glucose, and lipids) that could indicate presence of
different metabolic disorders. Two of the women were
rejected, and the remaining eight completed a VO2max
test on a stationary bike (Bikerace HC600; Technogym,
Gambettola, Italy). During the VO2max test, blood pressure
(Baumanometer, 300 model; W. A. Baum Co. Inc., New
York, NY), electrocardiogram (Nihon Kohden Electrocardiograph ECG-9329K; Tokyo, Japan), and oxygen uptake
(Innovision A/S model AMIS2001; Odense, Denmark) were
measured continuously until voluntary fatigue. Two participants were disqualified for irregular electrocardiograms, so
a total of six healthy early postmenopausal women were
included in the study. The study was approved by the local
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1
POSTEXERCISE NUTRIENTS IN AGING WOMEN
1213
variances in energy expenditure. Adequate protein intake
was set to 0.8 g/kg/day [lowest daily recommendation for
adults with a limited level of physical activity (16)].
Ethical Committee of Copenhagen (KF) 11-066/01 and, in
accordance with the Declaration of Helsinki, the study
protocol, purpose, and possible risks were explained to each
participant before their written consent was obtained.
Dietary Control
Participants completed a weighed-food record on 4
nonconsecutive days prior to the first trial. The food
recordings were analyzed on Ankerhus software (Winfood,
version 2.0; Ankerhus, Denmark) for daily energy and
protein intake. The recorded amount of daily energy intake
was compared to an estimation of adequate energy intake,
which was calculated by multiplying an estimated value of
the basal metabolic rate from the Harris-Benedict equation
with an activity factor set to 1.6 (16). Adequate energy
intake was set to .75% of calculated energy intake taking
both the accuracy of the Harris-Benedict equation (17) and
activity factor into consideration as well as individual
Analytical Procedures
The arterial–venous blood samples used for analysis of
amino acid concentration, amino acid enrichment, and
hormones were collected into 15% EDTA tubes (Vacutainer
Systems, Plymouth, U.K.), spun at 5000 rpm for 15 minutes
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Figure 1. Arterial plasma concentration of phenylalanine (Phe) (A) and
essential amino acids (EAA) (B). Values are mean; bars are standard error of the
mean (SEM). Squares represent the protein and carbohydrate (PC) group;
triangles represent the placebo (NON) group. ### denotes interaction ( p ,
.0001) in a two-way analysis of variance with repeated measures analysis.
Experimental Design and Protocol
Each participant was tested twice, at two different days,
separated by at least a 4-week ‘‘wash-out’’ period, using
a double-blinded randomized crossover design with supplement administration of either 10 grams of protein (soy and
milk protein), 31 grams of carbohydrate (dextrose), and 1
gram of fat, a total of 725 kJ (PC), or an equal-tasting placebo
product containing 6 grams of carbohydrate providing 100 kJ
(NON) (Otsuka Pharmaceuticals Co, Ltd., Saga, Japan)
(Figure 1). At least 1 week prior to the first trial each
participant determined their 10 repetition maximum (RM)
for each exercise and was familiarized to the protocol. The
exercise protocol consisted of three exercises: leg-press
exercise in supine position with feet high, resulting in a squatlike exercise (3 sets times 10 repetitions at 10 RM); leg-press
exercise with a low foot position, isolating quadriceps as the
prime-mover (4 sets times 10 repetitions at 10 RM); and knee
extension (4 sets times 10 repetitions at 10 RM). Two days
prior to each trial the participants were instructed to avoid
strenuous physical activities and caffeine, and to eat their
normal meals. Participants fasted from 10 PM the previous
night, with water allowed ad libitum during both fasting and
the trial. The participants arrived in the laboratory at 7:30 AM
by car and then rested in a bed. A catheter was inserted in the
antecubital vein, and a basal blood sample was drawn. At
8:00 AM (120 minutes), a primed (3 lmol/kg), constant
(0.05 lmol/kg/min) infusion of L-[ring-2H5]phenylalanine
(98% enriched; Cambridge Isotope Laboratories, Andover,
MA) was initiated with a target tracer-to-tracee ratio of 5%–
10% in arterial blood. Tracers were dissolved in sterile 0.9%
saline solutions and filtered through a 0.2-lm sterile disposable filter before infusion. At 9:30 AM (30 minutes), the
resistance exercise session was started. After 5 minutes of
warming up on a cycle ergometer (cadence .60 rpm), the
exercise protocol was conducted. All sets were conducted
as fast as possible, with an interval of 2 minutes between
each set with participants remaining passive on the training
equipment. Immediately after the completion of training, the participants consumed the supplementation within
1 minute. Supplement ingestion was designated as time zero
(0 minutes).
A catheter was then inserted following local anesthetic in
the right femoral artery using the Seldinger technique (18),
with another catheter placed retrogradely in the femoral vein
of the same leg. At 30, 60, 90, 120, 180, and 240 minutes,
arterial and venous blood samples were drawn from the
femoral catheters. Simultaneously, blood flow was determined in the contralateral femoral artery just above the
bifurcation of the femoral artery by the ultrasound Doppler
(Siemens, Ballerup, Denmark) technique (19).
HOLM ET AL.
1214
Calculations
Protein kinetics.—Net leg balance of phenylalanine was
derived from an equation based on the Fick Principle:
Net balance ¼ ð½phea ½phev Þ BF
½1;
where [phe]a and [phe]v are blood concentration of
phenylalanine in arterial and venous blood, respectively,
and BF is the blood flow supplying the limb. A positive
value denotes net thigh uptake, and a negative value denotes
net thigh release of the specific substrate, here phenylalanine, which is neither synthesized nor metabolized in the
leg. It is assumed that muscle protein turnover primarily
accounts for the leg metabolism of phenylalanine.
Because the real precursor pool, aminoacylated tRNA, is
not determined by this model, we have chosen an approach
that does not attempt to estimate real kinetic values (22), but
that asserts that the irreversible loss of tracee (Rate of
disappearance, Rd) into the muscle-bed is estimated from
the fractional extraction (FE) of its tracer from the blood:
FE ¼ ½ð½phea Ea Þ ð½phev Ev Þ ð½phea Ea Þ
1
½2;
where Ea and Ev are the phenylalanine enrichment in arterial
and venous blood. Thus:
Rd ¼ FE Ea BF
½3
Using this approach, we find that the Rd value refers to the
net amount of tracee and tracer disappearing from the
arterial side into muscle tissue. This is only part of the real
precursor, because recycling of tracee, directly as a product
from breakdown to a precursor for synthesis, may take place
intracellularly. Therefore, Rd is an underestimation of the
real synthetic rate (22).
Rate of appearance (Ra) of tracee into the blood now can
be calculated by subtracting the NB from Rd:
Ra ¼ Rd NB
½4
Similarly, the Ra value represents the net amount of
tracee that makes it into the blood (22). This is less than the
total rate of production, due to recycling as described above.
Hence, Rd is an underestimation of protein breakdown as
well (22).
Plasma phenylalanine concentrations are corrected to
whole blood values by the hematocrit for calculation of NB,
Rd, and Ra:
Blood concentration ¼ ½pheplasma ð100 hct%Þ 1001
½5;
where [phe]plasma is the phenylalanine concentration in
plasma, and hct% is the hematocrit as a percentage.
Statistical analysis.—Data are expressed as means 6
standard error of the mean. The effect of supplementation
over time was evaluated by a two-way analysis of variance
with repeated measures. A Bonferroni post hoc test was
used to determine pairwise differences at individual time
points when significant group interaction appeared. A t test
was used to compare values to zero (dependent). Statistical
significance was set at p , .05. Analyses were completed
with Prism 4.0 (GraphPad Software, San Diego, CA).
RESULTS
Participant Characteristics
The physical fitness (VO2max) averaged 30.1 6 1.9 ml/kg/
min and body mass index 22.9 6 1.4 kg/m2, which are fairly
normal for individuals at this age. One participant exercised
regularly on either cycle ergometer or step-machine, whereas
two others used a bike as daily transportation. The three
remaining participants refrained from exercise except activity
necessary during everyday life. Mean age of the women was
56 6 1.1 years, and they averaged 6.2 6 0.7 years since their
last menstrual cycle. All participants had a plasma estradiol
concentration below 0.10 nmol/L, indicating a ceased
ovarian production of estradiol. Average daily energy intake
was 9532 6 749 kJ, which corresponded to the estimated
daily need of 8751 6 239 kJ. The average recorded daily
protein intake was 73 6 6 g, corresponding to values within
the range 0.9–1.7 g protein/kg body mass, which presumably
is a sufficient amount of protein for this group of individuals
to remain weight stable.
Amino Acid Concentration, Enrichment,
and Blood Flow
Between trials there was an interaction effect ( p , .0001)
for arterial phenylalanine concentration (Figure 1A) and
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at 48C, and immediately stored at 808C for later analysis.
Glucose and hematocrit samples were collected as whole
blood in lithium–heparin and analyzed immediately on an
ABL-700 series apparatus (Radiometer Medical A/S,
Copenhagen, Denmark). Insulin plasma concentration was
measured by an enzyme-linked immunosorbent assay
(ELISA) kit (DAKO, Glostrup, Denmark). Estradiol concentrations were analyzed from basal blood samples after 8
hours of fasting using a competitive immunoassay (Immulite
2000; Diagnostic Products Corporation, Los Angeles, CA).
Amino acid concentrations were determined from plasma
samples (200 ll), which were deproteinized with 200 ll of
3% sulfosalicylic acid. The supernatant was assayed with an
amino acid analyzer (L-8500; Hitachi, Tokyo, Japan) with
S-(2-aminoethyl)-L-cysteine used as an internal standard.
Plasma phenylalanine enrichment was determined from
plasma samples as phenyl isothiocyanate (PICT) derivates
(Fluka Chemie GmbH, Munich, Germany) by liquid
chromatography–mass spectrometry (Finnigan AQA, Manchester, U.K.) performed essentially as described elsewhere
(20,21). After centrifugation (10,000 rpm for 4 minutes) of
the plasma, 100 ll was mixed with 100 ll of internal
standard (Norleucine, 98% enriched; Cambridge Isotope
Laboratories, Andover, MA) in centrifugal filter devices
(Ultrafree-MC; Millipore Corporation, Billerica, MA) and
centrifuged for 45 minutes at 15,000 rpm. Coupling buffer
(methanol/water/triethylamine, 2:2:1) and PICT derivatization solution (triethylamine/water/PICT/methanol, 1:1:1:7)
were applied separated by N2-drying. Finally, 100 ll of
ammonium acetate buffer was applied.
POSTEXERCISE NUTRIENTS IN AGING WOMEN
1215
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Figure 2. Phenylalanine tracer enrichment in arterial plasma, n ¼ 4. Values
are mean; bars are standard error of the mean (SEM). Squares represent the
protein and carbohydrate (PC) group; triangles represent the placebo (NON)
group. ### denotes time effect ( p , .001) in a two-way analysis of variance
with repeated measures analysis. No difference appeared between trials.
essential amino acid concentration (Figure 1B). However,
no difference in arterial phenylalanine enrichment was
apparent between trials, but there was a time effect ( p ,
.001) (Figure 2). Similarly, blood flow changed ( p , .05)
over time from rest till 240 minutes with no difference
between trials (Figure 3).
Protein Kinetics
Phenylalanine net balance values changed over time ( p ,
.001) and were elevated in the PC group compared to the
NON group ( p , .001) (Figure 4A). Net balance values in
the PC group were not different from zero ( p .05) in contrast
to values in the NON group, which were less than zero ( p ,
.001).
Figure 3. Blood flow in femoral arterial vessel. Values are mean; bars are
standard error of the mean (SEM). Squares represent the protein and
carbohydrate (PC) group; triangles represent the placebo (NON) group. #
denotes time effect ( p , .05) in a two-way analysis of variance with repeated
measures analysis. No difference appeared between trials.
Figure 4. Net Balance of phenylalanine (A), phenylalanine rate of
disappearance (Rd) an estimate of protein synthesis (B), and phenylalanine rate
of appearance (Ra) an estimate of protein breakdown (C). Values are mean; bars
are standard error of the mean (SEM). Squares represent the protein and
carbohydrate (PC) group; triangles represent the placebo (NON) group. ###
denotes time effect ( p , .001) and *** denotes supplementation effect ( p ,
.001) in a two-way analysis of variance with repeated measures analysis.
1216
HOLM ET AL.
Glucose and Insulin
There was an interaction ( p , .001) between trials for
arterial glucose concentration (data not shown) with most
pronounced elevations at 60 and 90 minutes. Mean venous
insulin concentration rose threefold in the PC group from
fasting levels at rest to peak values at 60 minutes
(interaction p , .0001) (Figure 5).
DISCUSSION
The present study demonstrated that ingestion of protein
and carbohydrate immediately after a resistance exercise
session elevated the net protein balance across the trained
leg in middle-aged, early postmenopausal women when
compared to placebo intake after resistance exercise.
However, the response was attenuated when compared to
previously published data on young individuals.
Attenuated Response to Nutrients Following Exercise
in Elderly Persons
Until now no studies have used stable isotopic tracers and
net balance to examine the acute response to the combination
of exercise and nutrition in the elderly population. A previous
study on elderly persons demonstrated greater muscle
hypertrophy when supplementation is taken immediately
following exercise compared to 2 hours after (23), indicating
that timing of nutrient intake is important in older individuals.
This finding is similar to those in studies of young persons
that find that resistance exercise increases muscle protein
turnover but that nutrient intake is necessary to take
advantage of the full anabolic processes following resistance
exercise (11–13), as net balance remains negative when
fasting is sustained (6,11–13).
Our results show that, in early postmenopausal women,
resistance exercise does not increase net protein balance to
positive values when fasting conditions are maintained
(Figure 4A). However, with ingestion of 10 grams of protein
and 31 grams of carbohydrate, protein balance is elevated and
equals zero for up to 4 hours after exercise. Previous data
from young persons indicate that ingestion of a comparable
nutrient composition (6 grams of essential amino acids and
35 grams of glucose) after resistance exercise enhances net
protein balance to positive values (12,24). Therefore, our
results indicate that early postmenopausal women demonstrated an impaired, although elevated, responsiveness to
nutrient intake. Although young participants were not
compared to old participants in this investigation, the
impaired responsiveness to mixed nutritional intake may
help explain reduced lean body mass with increasing age.
Figure 5. Venous insulin concentrations. Values are mean, bars are standard
error of the mean (SEM). Squares represent the protein and carbohydrate (PC)
group; triangles represent the placebo (NON) group. ### denotes interaction
( p , .0001) in a two-way analysis of variance with repeated measures analysis.
We believe that previously published results in young and
old persons lend support to the idea of an impaired nutritional
response. First, it has been demonstrated that aging muscle
retains acute responsiveness (i.e., increased turnover) to
exercise (7,25) and retains ability to hypertrophy during
long-term training (23,26,27), and that mixed muscle and
myofibrillar protein synthesis rates after exercise are
comparable to those of young individuals (7,25). Second,
some degree of insulin resistance, which is frequently seen
among elderly persons (28), may result in diminished
nutrient responsiveness in old persons as compared to
younger persons. The role of insulin must be considered
because mixed meals (as in our study), as opposed to proteinonly meals, are more representative of daily food intake.
Finally, although the overall time-dependent response to
amino-acid ingestion may not vary between older and
younger individuals, the anabolic response in the first hour
following ingestion is attenuated in the older individuals (9).
Such attenuation in the first hour following ingestion has
been attributed to a greater first-pass extraction in the gut of
older persons (9,29), which may be an effect of insulin
resistance (30). The rapid increase in plasma and intracellular
amino acid concentration could be crucial in the light of the
proposed ‘‘critical period’’ of nutrient ingestion following
exercise (23).
Therefore, it appears that responsiveness to exercise
(acute and chronic) is maintained, but attenuated sensitivity
to insulin and an attenuated initial increase in plasma amino
acids may account for the attenuated net balance response.
However, it is important to note that despite the attenuated
response (compared to the positive net balance in young
participants) the early postmenopausal women still benefited
from the addition of nutrient ingestion following exercise.
Methodological Considerations
We chose to use the two-pool model for calculation of
substrate kinetics. Fundamentally, isotopic as well as
physiological steady state is a prerequisite for the use of
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Because isotopic steady state is required for calculations
of amino-acid kinetics, two of the participants were
eliminated from this calculation due to wave-like fluctuations in arterial phenylalanine enrichment over time. Hence,
kinetic data are calculated for four participants, who had
linear, slightly elevating enrichment curves, indicating
isotopic steady state at each individual measure point. On
n ¼ 4, no statistical differences appeared for Rd and Ra
(Figure 4, B and C). However, p values for interaction were
.12 and .20 for Rd and Ra, respectively.
POSTEXERCISE NUTRIENTS IN AGING WOMEN
Amino Acid Kinetics and Net Balance
As described above, it is known that the two-pool model
underestimates the real values for protein synthesis and
breakdown (22). However, in theory both variables are
underestimated by the same value, i.e., the rate of
intracellular recycling of tracee. Hence, the mutual relations
between variables calculated with this approach should
correspond to the real kinetic values.
Although it must be considered that the phenylalanine
kinetics are from an n of 4, the results are discussed in
support of our net balance measurements. Ingestion of
protein and carbohydrate tended ( p ¼ .12) to elevate Rd
(protein synthesis) in the exercised limb during the period
in which amino acid availability was increased. This
availability-dependent effect of amino acids, and especially
essential amino acids on protein synthesis, is previously
reported in young persons (12,24,33). In a recent study on
resting elderly persons (9) a similar time course for arterial
phenylalanine concentration and net balance was reported
following oral ingestion of 15 grams of amino acids. As
discussed above, compared to the response observed in
younger individuals, the aged individuals seem to have
a slower and more prolonged response (9). Because in the
present study net balance is not different from zero in the
initial period following ingestion, early postmenopausal
women may respond more similarly to elderly participants
than to younger participants.
In the present study, in which carbohydrate and protein
was ingested, it was expected that Ra (protein breakdown)
would be diminished (15). After exercise and PC, phenylalanine Ra peaked at 30 minutes and was equal to NON by 60
minutes. However, the Rd increased sufficiently to maintain
net balance not different to zero, whereas in the NON group,
Ra exceeded Rd resulting in negative net balance throughout
the period. In the PC group, insulin concentration peaked at
60 minutes after ingestion. Because insulin is known to have
a potent, postponed anticatabolic effect (34), it is interesting
that the decreased Ra at 60 minutes preceded the insulin peak
at 60 minutes, and that no significant change in Ra in the
period following the insulin peak appeared. As discussed
above, a decreased insulin response (either centrally or
peripherally) could account for the lack of significant change
in Ra and the attenuated net balance response in the early
postmenopausal women compared to data from younger
participants. However, as with net balance, it is important
to realize that there was indeed a positive effect of PC ingestion compared to NON following exercise.
Conclusion
The present study demonstrated that early postmenopausal
women did benefit acutely from ingestion of protein and
carbohydrate immediately after a resistance exercise session
to increase skeletal muscle protein accretion. This finding
provides the basis for a long-term effect of such nutrient
intake in combination with exercise training in postmenopausal women in counteracting muscle loss with aging.
ACKNOWLEDGMENTS
We thank the participants for their attendance in this study, and we are
grateful for the technical assistance of Annie Høj, Birgitte Lillethorup, AnnMarie Sederstrøm, and Ann-Christina Henriksen during the studies and
with analysis of samples.
The study was supported by Otsuka Pharmaceuticals Co. Ltd.,
Saga, Japan.
Address correspondence to Lars Holm, Institute of Sports Medicine,
Copenhagen, Bld. 8 1st Bispebjerg Bakke 23, 2400 Copenhagen NV,
Denmark. E-mail: lh17@bbh.hosp.dk
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Received June 14, 2004
Accepted July 13, 2004
Decision Editor: John E. Morley, MB, BCh
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